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  • Merging microscopy methods sharpens brain imaging

BioPhotonics
Nov 2012

SESTO FIORENTINO, Italy – Combining the best features of two microscopy methods yields images 100 percent sharper than those acquired through conventional light-sheet-based microscopy (LSM).

LSM, also known as single-plane illumination microscopy, uses a laser beam, narrowed to just a few microns across, to illuminate a biological sample from the side – instead of from above or below – with a thin sheet of light. A lens is then used to focus the fluorescence radiated from the sample upward to be captured by a digital camera.

A drawback of the method is that it enables only a portion of the sample to be imaged at a time. Rotating the sample, as well as raising and lowering the illumination plane, produces a series of two-dimensional sectional views, or “slices,” that can be pieced together to yield a 3-D map of a whole organism or any of its organs or systems.


Purkinje cells from a mouse cerebellum imaged (a) with light-sheet microscopy and (b) with the significantly higher contrast provided by confocal light-sheet microscopy. The scale bar at the bottom is 100 µm across. Courtesy of Optics Express/European Laboratory for Non-Linear Spectroscopy, University of Florence, Italy.


The method developed by a team in Italy allows high-speed, single-plane images of multiple sections of a sample to be taken while also eliminating the scattered background light that causes blurriness.

The integrated LSM/confocal microscopy technique, called confocal light-sheet microscopy (CLSM), uses a filter to remove photons that stray from the thin sheet’s single plane.

The combined systems “filtered the scattered photons that were emitted and recovered the normally lost image contrast in real time without the need for multiple acquisitions or any postprocessing of the acquired data,” said Francesco Pavone, leader of a collaborative team comprising six Italian research agencies, and an author of the paper describing the advance.

Researchers have tried to map the brain’s billionfold neural network with conventional LSM, but while the technique yields high-resolution views of tissue excised from mouse brains and those fixed in position, it cannot obtain whole-brain images. Whole-brain samples scatter the emitted light and create background fluorescence that reduces contrast and blurs the perceived image, an aberration that makes it difficult to resolve and reconstruct the entire neuronal network with high contrast.


Purkinje cells’ micron-scale neuroanatomy in the whole cerebellum. (a) 3-D volume rendering of a PND-10 L7-GFP mouse cerebellum. The superimposed planes refer to transverse (red), sagittal (green) and coronal (blue) digital sections shown in panels (b), (c) and (d) respectively. (b-d) Maximum-intensity projections of 40-µm-thick slabs. Scale bars, 1 mm. (e, f) 10× magnification of the regions highlighted by the yellow boxes in panels (b) and (d). The look-up table saturates 2 percent of pixels for better visibility. Courtesy of Optics Express.


The researchers say the new technique proved superior over conventional LSM microscopy and a variation of LSM that requires redundant slices and postprocessing to remove scattered light when used to view three samples of the mouse brain: the hippocampus, the cerebellum and the whole brain. They also used CLSM to map the micron-scale neuroanatomy of mouse Purkinje cells – large neurons found in the cerebellum – and to trace an entire brain’s neuronal projections.

This is the first time that a fluorescent mouse brain has been imaged in its entirety with such clarity, said Ludovico Silvestri, a member of the research team. The technique also could be extended to the human brain, in principle, but it will first be necessary to overcome the problem of staining fixed tissue fluorescently.

“The high-contrast fluorescence and fast acquisition assured by CLSM may represent a powerful tool to help neuroscientists navigate through the neuronal pathways of the brain,” Pavone said. “Although this study was focused on brain imaging, we believe that CLSM also is ideally suited to explore – at micron-scale resolution – the anatomy of different specimens, such as murine organs, embryos and flies.”

CLSM also has been used to image mouse models of several diseases, including autism and ischemic stroke, Silvestri said. “We hope that the whole-brain tomographies we can obtain will one day provide insights into the mechanisms of these and other brain disorders.”

The study appeared in The Optical Society’s open-access journal Optics Express (doi: 10.1364/oe.20.020582).


GLOSSARY
digital camera
A camera that converts a collected image into pixels that are black or white digital or shades of gray. The digital data may then be manipulated to enhance or otherwise modify the resulting viewed image.
fluorescence
The emission of light or other electromagnetic radiation of longer wavelengths by a substance as a result of the absorption of some other radiation of shorter wavelengths, provided the emission continues only as long as the stimulus producing it is maintained. In other words, fluorescence is the luminescence that persists for less than about 10-8 s after excitation.
lens
A transparent optical component consisting of one or more pieces of optical glass with surfaces so curved (usually spherical) that they serve to converge or diverge the transmitted rays from an object, thus forming a real or virtual image of that object.
photonics
The technology of generating and harnessing light and other forms of radiant energy whose quantum unit is the photon. The science includes light emission, transmission, deflection, amplification and detection by optical components and instruments, lasers and other light sources, fiber optics, electro-optical instrumentation, related hardware and electronics, and sophisticated systems. The range of applications of photonics extends from energy generation to detection to communications and...
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